Maintenance of the correct genetic information is crucial for all living organisms. Mutations are the primary cause of hereditary diseases, as well as cancer, and may also be involved in aging. 80 to 90% of all human cancers are ultimately due to DNA damage. Different repair mechanisms have evolved to protect the genome. Nucleotide excision repair (NER) is well known for the removal of bulky DNA lesions and is unique in its versatility to repair a broad substrate range of DNA lesions. In humans, NER is the major repair mechanism to protect DNA from damage induced by ultraviolet light. The phenotypic consequences of defective genes involved in NER are apparent in three severe diseases: xeroderma pigmentosum, Cockayne's syndrome and trichothiodystrophy. The overall goals of this project are to understand the fundamental mechanisms of nucleotide excision repair by a series of studies involving the bacterial UvrABC NER machinery. In these studies specific proteins and complexes involved in nucleotide excision repair will be characterized by a combination of biochemical, crystallographic and molecular biology experiments. The studies described in this project will verify three hypotheses: (1) The bacterial NER protein UvrB recognizes damage by intercalating a b-hairpin between the DNA duplex and forms tight interactions with the undamaged strand only after formation of the pre-incision complex. (2) The lesion containing strand is mainly held in place by base stacking interactions and is freely accessible for recognition by UvrC, the endonuclease, which is responsible for the excision process. (3) A conformational change takes place in either UvrB and/or UvrC to allow the sequential process in which 3' incision precedes 5' incision. The proposed studies are divided into four specific aims: (1) Characterization of the UvrA/UvrB interaction and identification of DNA binding sites on UvrB. (2) The pre-incision complex: Structural characterization of the UvrB.DNA pre-incision complex. (3) Characterization of UvrC in the absence of UvrB and DNA: Determination of the three-dimensional structure of UvrC prior to binding to UvrB and DNA. (4) Analysis of the UvrB.UvrC.DNA complex: The proposed studies will delineate the roles of the individual proteins and their complexes formed in the process of NER.